Posts Tagged ‘DNA ladder’

Gel electrophoresis is a core technique used in molecular biology laboratories. The gels used for electrophoresis are almost always agarose which is a polysaccharide purified from agar-agar. Agar-agar is obtained from red algae and contains a variety of impurities along with the agarose. Agar-agar is often used as a substitute for gelatin in vegetarian dishes and is used to make some cool culinary creations. Pure agarose is obtained by separating it from agar-agar. I have heard from variety of sources that agar-agar could be used in place of agarose for gel electrophoresis of DNA. Given that agarose costs around $1.00 per gram and agar-agar costs around $0.05 per gram I thought it was worthwhile to check the efficacy of agar-agar. My results show that agar-agar is not an acceptable substitute for agarose (see the image above).

For more details and thoughts read the rest of this post.

Cleaning the Agar-agar

The night before I ran the experiment I decided that it would be nice if I tried to “clean” the agar-agar of some impurities and run this as an additional treatment group. Since I did this last minute I did not completely think my plan of attack through and actually made the agar-agar worse. I used 50% isopropanol and distilled water to rinse the agar-agar through a coffee filter.

After running the experiment and seeing my results I did the research I should have done prior to the experiment. A quick google search showed me a technique for purifying agarose from agar-agar using propylene glycol. Click here for the site in question.

Failed Purification Protocol:

Weighed out 11.7g agar-agar into a coffee filter (2 thick).

Saturated agar with distilled H2O (dH2O).

Poured 100mL of dH2O through the slurry and allowed it to drain.

Stirred slurry with a spatula.

Pourd 100mL of dH2O through the slurry and allowed it to drain.

Stirred slurry with a spatula.

Poured 100mL of 50% isoproanol into the slurry.

Stirred slurry with a spatula.

Once the majority of fluid drained out I placed the agar-agar and coffee filter into an oven at 90C for 5 hours to dry.

Experiment Parameters

1.2% gel

TAE Buffer

2.5 hours

70 volts

Biotium GelRed Stain (used as a precast additive)

Promega 1kb DNA Ladder (left lane)

NEB 2-Log DNA Ladder (right lane)

Pictures and Comments

Agarose gel atop the UV transilluminator.

I had minor issues taking pictures of the gels because too much light was reaching the camera lens so I made a aluminum foil frame to block out extra light.

All 3 gels deformed on the end closest to the (-) electrode and I am not sure why. I suspected temperature at first but it subjectively seemed constant throughout the gel rig. Next I thought it was the gels positioning, however when checked I found each end to be equally distant from both electrodes.

Compare the picture above to the reference gel below (in black and white). This reference gel was the first gel I ran with my gel rig and it ran for 80min at 70V using the same TAE buffer used in this experiment and there was no deformation of the gels wells. The two differences between the reference gel and the newest gels were time and staining compound (2.5 hours vs. 80min and ethidium bromide vs. GelRed). If this problem persist I will have to run a bunch of gels to figure this out.

See blurb above for information about this reference gel.

All 3 experimental gels on the UV transilluminator. From left to right; agar-agar, cleaned agar-agar, and agarose.

Conclusions

Based on my results I do not recommend purchasing agar-agar for gel electrophoresis. The only exception I would make is if the intent is to use propylene glycol to separate the agarose from the agar-agar (click here for agarose separation with propylene glycol). Keep in mind that there could be nuclease and cation contamination in the propylene glycol purified agarose.

While agarose is a lot more expensive than agar-agar, it is not expensive relative to everything else needed for conducting molecular biology at home. For the sake of simplicity, reliability, and time I intend on using only agarose going forward.

What is next

My next post will compare running buffers between gels – specifically molecular biology grade Tris-Acetate-EDTA (TAE buffer), Molecular Bio grade sodium borate buffer (SB Buffer), and sodium borate buffer made from borax and cockroach poison (boric acid). If there is any simple household substances you want me to try as a running buffer, let me know. I have a lot of school and work projects going on so this upcoming post may be a few weeks out.

It took much longer for me to get a round to testing out my gel electrophoresis equipment than I thought it would. For now I have merely got it to work. Next I will try and fine tune it to increase the quality of the gels. More on that below.

This isn’t the most informative post but I was kind of frustrated by a lack of information when I was troubleshooting so I figured I would throw some data out and hope it helps someone.

Note: The cameras on my phone and iPad both captured more wavelengths of light than I could see, so these images look worse than the gel actually is.

The image above is what the first two gels looked like – no fluorescence at all. I still do not know for sure why they failed but I narrowed it down to either the composition of the DNA ladder or the staining method.

My set-up worked when I used GelRed in the molten agar-agar and composed the DNA ladder per the manufacturers instructions. I used 5-10uL of ladder and 1-2uL of loading buffer on the failed gels whereas I used 1uL of ladder, 4uL H2O, and 1uL loading buffer on the successful gel. During the failed gels I tried to use the 3x post-electrophoresis stain procedure with staining times between 0.5-1.5 hours – all with no luck. Obviously changing two variables at once confounds the results – but at least I have a baseline now.

The setup

This was the gel box I built and used. You can read my instructions for how to build it here.

Muh lab.

This is ~90% of my apartment lab.

My next goal is to work out how to fine tune the procedure. I am going to compare the gel quality in cases where I use reagent grade vs. industrial and food grade chemicals. I am hoping that borax (sodium tetraborate) and roach poison (boric acid) buffer will work as good as its reagent grade cousin.